The present invention relates to the specific interactions of low molecular weight compounds with RNA. More particularly, the present invention relates to compositions, methods and kits for identifying compounds that interfere with RNA-protein or RNA-ligand interactions and thereby interfere with RNA function.
In most biological systems, the maturation, transport, stability and expression of RNA is closely regulated by the interactions between highly conserved regulatory RNA sequences and proteins. In many circumstances it is desirable to develop drugs that bind RNA at sites of regulatory protein binding and act as competitive inhibitors of the RNA-protein interaction. These types of drugs have potential applications in a wide range of diseases including viral, bacterial and fungal infections and chronic diseases such as cancer and autoimmune disease.
1. RNA Targets for Drug Discovery
Although RNA is often referred to as being single stranded and unstructured, most biologically active RNA molecules actually fold back on themselves to create a wide variety of structures. In RNA structures, the secondary structure is energetically the largest contributor to the overall three-dimensional fold. The main secondary structure element in large RNA molecules is the RNA double helix built by Watson-Crick base pairings between two regions of the RNA polynucleotide. The helical elements in RNA are typically interrupted by bulges and internal loops. In addition to disruptions of the helical structures, biologically active RNA molecules typically contain specialized loop sequences that create stable bends in RNA. Associations between single stranded regions and between single stranded regions and double helices lead to structural elements creating tertiary structures. Many tertiary structural elements in RNA form recurrent motifs, such as xe2x80x9cpseudoknotsxe2x80x9d created by the interactions of pairs of loop structures. Additional tertiary structure elements such as base triples are also commonly found in large RNA structures.
Regulatory proteins seldom target fully-double stranded regions of RNA, which like A-form DNA forms a comparatively rigid structure in which most functional groups that distinguish between the bases are inaccessible. Instead, RNA-binding proteins generally target functional groups where bulges, loops and hairpins disrupt the regular RNA helix and open up the major groove for interaction with protein side chains. Regulatory proteins usually interact with these bulges, loops and hairpins that disrupt the regular RNA helix. These secondary structure elements provide the structural diversity needed for protein recognition as well as for drug development.
2. RNA Targets in HIV
A great deal of the present knowledge about the structure of RNA targets and their recognition by RNA binding proteins has come from studies of two human immunodeficiency virus (HIV) regulatory proteins Tat, the trans-activator protein and Rev, the regulator of virion expression. The two proteins play complementary roles in the virus life cycle. Tat stimulates transcription from the viral long terminal repeat (LTR), whereas Rev is required for the efficient export from the nucleus of the late mRNAs encoding the structural proteins of the virus.
Tat and Rev both exert their effects through specific cis-acting viral RNA regulatory sequences. Tat activity requires the trans-activation-responsive region (TAR), an RNA regulatory element of 59 nucleotides (nt) located immediately downstream of the initiation site for transcription (Cullen, 1990; Karn et al., 1995). Because of its position in the HIV genome, each viral mRNA carries a copy of TAR at its 5xe2x80x2-end.
TAR RNA forms a highly stable, nuclease-resistant, stem-loop structure. Point mutations, which disrupt base-pairing in the upper TAR RNA stem invariably, abolish Tat-activated transcription. In 1989 Dingwall et al. provided the first demonstration that recombinant Tat expressed in Escherichia coli could bind specifically to bases in the stem of the TAR RNA (Dingwall et al., 1989). The binding site for Tat includes a U-rich trinucleotide bulge found in the upper stem of TAR RNA.
Extensive mutagenesis, chemical probing and peptide binding studies have defined the key elements required for TAR recognition by Tat and have shown that the Tat binding site surrounds a UCU bulge located near the apex of TAR (Calnan et al., 1991a; Weeks and Crothers, 1991; Delling et al., 1992; Churcher et al., 1993; Hamy et al., 1993; WO92/02228). Tat interacts with the first uridine of the bulge, U23, while the other residues in the bulge act predominantly as spacers and may be replaced by other nucleotides, or even by non-nucleotide linkers (Churcher et al., 1993). Tat recognition requires two base-pairs in the stem above the U-rich bulge, G26xc2x7C39 and A27xc2x7U38 (Weeks and Crothers, 1991; Delling et al., 1992; Churcher et al., 1993). Two base pairs below the bulge, A22xc2x7U40 and G21xc2x7C41, also make significant contributions to Tat binding. Critical phosphate contacts involve phosphates P21, P22, and P40, which are located below the bulge on both strands (Calnan et al., 1991b; Hamy et al., 1993; Pritchard et al., 1994).
In addition to binding the Tat protein, TAR is able to form complexes with short peptides containing an arginine-rich sequence derived from residues 48 to 57 of Tat (Cordingley et al., 1990; Weeks et al., 1990; Calnan et al., 1991a; Weeks and Crothers, 1991; Churcher et al., 1993). However, compared to the Tat protein, short arginine-rich peptides bind with reduced affinity and, in certain cases, also have a significantly reduced binding specificity (Churcher et al., 1993). For example, short basic region peptides (such as the ADP-3 peptide, residues 48 to 72) show less than a 2-fold difference in affinity between wild-type TAR RNA and TAR sequences carrying the mutations U23xe2x86x92C and G26xc2x7C39xe2x86x92Cxc2x7G mutations (Churcher et al., 1993). Similarly, some basic peptides display a high affinity for any duplex RNA sequences that carries one or more bulged residues. Monomeric complexes between peptides and TAR are only obtained when TAR is truncated and the competing binding sites normally present on the lower stem are removed (Weeks et al., 1990; Weeks and Crothers, 1991; Churcher et al., 1993). By contrast, peptides such as ADP-1 (residues 37 to 72, see FIG. 3) that contain residues from the conserved xe2x80x9ccorexe2x80x9d region of lentivirus trans-activators, have a higher affinity for TAR RNA and are able to discriminate between TAR RNA mutants with a specificity which more closely resembled that of the Tat protein itself (Churcher et al., 1993).
Recent NMR studies of TAR RNA demonstrate that the accessibility of the critical functional groups recognized by Tat and basic peptides is enhanced by rearrangement of the bulge region (Puglisi et al., 1992; Aboul-ela et al., 1995; Aboul-ela et al., 1996). This refolding process involves one of the arginine side chains present in the basic binding domain of the Tat protein (Puglisi et al., 1992; Aboul-ela et al., 1995). In the presence of the arginine, the stacking of the bulged residues U23 on A22 and C24 on U23 is disrupted and A22 becomes juxtaposed to G26. This creates a binding pocket where the guanidinium and (xe2x80x94NH groups of the arginine are placed within hydrogen-bonding distance of G26-N7 and U23-04, respectively. The conformational change in TAR RNA also repositions the P22, P23 and P40 phosphates, which provide energetically important contacts with Tat (Pritchard et al., 1994).
The Tat-TAR interaction therefore provides a clear example of the xe2x80x98indirect readoutxe2x80x99 of nucleic acid sequences through recognition of backbone phosphates. The importance of arginine binding pocket in TAR for Tat binding is confirmed by the observation that the mutations that produce the most severe reductions in TAR activity involve G26 and U23 and disrupt the intermolecular interactions that are responsible for the folding transition (Weeks and Crothers, 1991; Churcher et al., 1993).
Rev activity requires a second cis-acting sequence, called the Rev-response element (RRE) which is located within the env reading frame (Malim et al., 1989a). The RRE contains a series of stem loop structures protruding from a long central stem, Stem I (Malim et al., 1989b; Mann et al., 1994). Near the apex of Stem I is a high affinity binding site, which is recognized by a monomer of Rev protein with a Kdxcx9cnM (Bartel et al., 1991; Heaphy et al., 1991; Iwai et al., 1992; WO92/05195). The high affinity site is a purine-rich bubble stabilized by non-Watson-Crick Gxc2x7A and Gxc2x7G base pairs (Bartel et al., 1991; Iwai et al., 1992; Pritchard et al., 1994). The non-Watson-Crick base pairs, along with a bulged-out uridine nucleotide, open the major groove and permit the recognition of functional groups on the two base pairs either side of the bulged region (Iwai et al., 1992; Kjems et al., 1992; Pritchard et al., 1994). In addition to the base-specific contacts, phosphate contacts are made around the bubble and up to six nucleotides away from the bubble, towards the apex of the stem-loop (Iwai et al., 1992; Kjems et al., 1992; Pritchard et al., 1994).
Mutational analysis of the RRE has shown that the high affinity site is necessary, but not sufficient, for Rev activity in vivo (Malim et al., 1989b; Mann et al., 1994). The binding of a Rev monomer to the high affinity site nucleates the co-operative oligomerization of Rev protein along flanking RNA sites in Stem I (Heaphy et al., 1990; Heaphy et al., 1991; Malim and Cullen, 1991; Mann et al., 1994; Zemmel et al., 1996; WO97/39128). The simplest way to visualize this process is by gel mobility shift assays. These assays show that there is a progressive increase in the formation of the highest molecular complexes as the molar ratio of Rev to RRE RNA increases (Heaphy et al., 1990; Heaphy et al., 1991; Kjems et al., 1991; Malim and Cullen, 1991; Zemmel et al., 1996). Truncations of Stem I that do not affect the high affinity site, reduce Rev responses by removing secondary binding sites, with the longest truncations producing the greatest losses of activity (Mann et al., 1994). Similarly, mutations in the Rev protein that block oligomerization produce an inactive protein (Malim and Cullen, 1991). These observations suggest that the RRE acts as a xe2x80x98molecular rheostatxe2x80x99 designed to detect Rev levels during the early stages of the HIV growth cycle (Mann et al., 1994).
Short basic peptides are also able to bind to the RRE high affinity binding site (Kjems et al., 1992; Battiste et al., 1994; Battiste et al., 1996; Jain and Belasco, 1996). Gel mobility shift assays have shown that basic peptides containing residues that have a propensity to form alpha-helices bind the RRE with enhanced affinity (Tan et al., 1993). Rev suppressor mutations that alleviated the deleterious effects of mutations in the RRE high affinity binding site all map to a single arginine-deficient face of a Rev alpha-helix, providing genetic evidence for direct contacts between specific Rev amino acids and RNA nucleotides in the RNA complex of Rev (Jain and Belasco, 1996).
Frankel et al. (WO94/29487) have described compositions of peptide analogues that mimic the RNA-binding domain of the native Rev protein and are able to bind to the HIV RRE with nanomolar affinity. All of these peptides have a propensity to form alpha-helices.
The structural basis for Rev binding to the RRE high affinity site has recently been revealed by NMR studies (Battiste et al., 1996; Peterson and Feigon, 1996; Ye et al., 1996). These studies demonstrate that the RRE high affinity site contains a large open major groove, which is able to accommodate an alpha-helical peptide. The phosphate backbone adjacent to the Gxc2x7G base pair undergoes a conformational rearrangement during peptide binding and adopts an unusual locally parallel-stranded orientation. This distortion results in an under twisting of the base pairs in the bulge region and an opening of the major groove by approximately 5 xc3x85. The Rev alpha helix appears to penetrate much more deeply into the major groove than is typical of DNA binding proteins. Several arginine side chains from the peptide make base-specific contacts, and an asparagine residue contacts the Gxc2x7A base pair.
3. RNA Mimics of Regulatory Protein Binding Sites
Small fragments of RNA are often able to fold into structures that mimic protein binding sites. Model RNAs that fold into the correct structures are able to bind regulatory proteins with similar affinity and specificity to the original RNA sequences and are commonly used as components in assays for RNA-protein interactions since their small size permits synthesis on a large scale either by chemical methods or by transcription from DNA templates.
Karn et al. (WO92/02228 and U.S. Pat. No. 5,821,046 (issued Oct. 13, 1998)) have described oligonucleotide stem-loop structures and duplexes that form analogues of the Tat binding site on TAR RNA. They have also disclosed an assay for identifying a compound that inhibits the binding of Tat protein to TAR RNA based on competition between the compound-to-be-tested and the Tat protein for binding to TAR RNA and its analogues.
The high affinity Rev binding site can also be mimicked by artificial stem-loop structures carrying fragments of the RRE (Bartel et al., 1991; Heaphy et al., 1991; Kjems et al., 1992; Pritchard et al., 1994). Karn et al. (WO92/05195 and U.S. Pat. No. 5,786,145 (issued Jul. 28, 1998)) have described compositions of oligonucleotides that, when folded, correspond to the high affinity site bound by the HIV Rev protein. Karn et al. (ibid.) also describe an assay for identifying compounds that inhibit Rev binding based on competition between the compound-to-be-tested and the Rev protein for binding to RRE RNA and its analogues.
A model RNA sequence for the aminoglycoside binding site on Escherichia coli 16S rRNA has been described by Purohit and Stern (1994, and U.S. Pat. No. 5,712,096 (issued Jan. 27, 1998). These RNA model sequences include a nucleic acid structure derived from the parental ribosomal RNA that is capable of binding to a ligand (such as the aminoglycoside) in the original RNA structure and a stabilizing sequence that provides the model RNA with a conformation that permits ligand binding that is substantially identical to the parental RNA ligand binding pattern.
4. Non-fluorescent Assays for RNA-protein Interactions
A critical step in the development of RNA-binding drugs is the development of simple and robust assays that are suitable for the high throughput screening of large compound libraries developed either by combinatorial synthesis traditional medicinal chemistry approaches, or from collections of natural products. The original assays for RNA-protein interactions involving the HIV regulatory proteins were based on filter binding (Churcher et al., 1993; Mei et al., 1997), gel retardation (Calnan et al., 1991a; Weeks and Crothers, 1991; Churcher et al., 1993; Hamy et al., 1993; Hamy et al., 1997; Mei et al., 1997), scintillation proximity (Mei et al., 1997) or electrospray mass spectroscopy (Sannes-Lowery et al., 1997). In all of these assays the free protein (or peptide mimic of the protein) or the free RNA is separated from the complex by physical means. For example, in the gel shift assay, the RNA is labeled by a radioactive or fluorescent group and the free RNA is separated from the complex by electrophoresis. In the filter binding assay, free RNA is separated from protein-bound RNA because of selective binding of the protein to nitrocellulose filter membranes, while in the scintillation proximity method, RNA is bound to the surface of a bead carrying scintillant and free peptide carrying a radioactive label is brought into contact with the bead because of its affinity for RNA. In the mass spectroscopy assay the peptide-RNA complex is ionized and separated by electrospray. Unfortunately none of these assays is ideal for high throughput screening, since they each require a large number of manipulations after the binding reactions have been set up.
5. Use of Fluorescent Probes to Measure Binding of Ligands to Nucleic Acids and Conformational Changes in Nucleic Acids
There are many different types of assays that measure the binding of ligands to nucleic acids, or conformational changes in nucleic acids and utilize fluorescence resonance energy transfer (FRET) to generate a signal. FRET is caused by a change in the distance separating a fluorescent donor group from an interacting resonance energy acceptor, either another fluorophore, a chromophore, or a quencher. Combinations of donor and acceptor moieties are known as xe2x80x9cFRET pairsxe2x80x9d. Efficient FRET interactions require that the absorption and emission spectra of the dye pairs have a high degree of overlap. FRET is also a distance-dependent interaction which is dependent on the inverse sixth power of the intermolecular separation, making it a sensitive measurement of molecular distances (Stryer, 1978 and Selvin, 1995).
One application of this technology is to measure incorporation of new bases for the purposes of genetic diagnostics. Chen and Kwok (1997) and Chen et al., (1997), demonstrated that a 5xe2x80x2 fluorescein-labeled primer can be extended with a dye-labeled dNTP and modified Taq DNA polymerase. The doubly-labeled DNA oligonucleotide can then be detected by measuring changes in fluorescence intensity.
A variety of assays that utilize FRET to detect hybridization between nucleic acids carrying fluorescent dyes are known. Tyagi and Kramer have described a method of xe2x80x9cmolecular beaconsxe2x80x9d (WO95/133399; WO97/39008; and Tyagai and Kramer, 1996) in which a fluorescently labeled DNA reporter is constructed that carries a pair of fluorescent dyes on its 3xe2x80x2 and 5xe2x80x2 ends, a target recognition signal, and arms surrounding the target recognition signal that can hybridize and form a hairpin structure with the target sequence as the single stranded region. In this conformation, the FRET dye pair on the reporter are brought into close proximity and quenched by FRET. When the reporter molecule hybridizes to a second DNA molecule that is complementary to the target recognition sequence, the stem of the reporter is disrupted and a conformation change is induced that results in an increase in fluorescence and/or a decrease in quenching.
Another method to detect hybridization between two DNA molecule that is based on FRET is described by Cardullo et al. (1988). This method utilizes a probe comprising a pair of compentary oligodeoxynucleotides, one of which contains a fluorophore on its 5xe2x80x2 end, and the other of which contains a complentary fluorophore on its 3xe2x80x2 end. Hybridization of the two probes brings the fluorescent groups into proximity and results in FRET. A related method is described by Heller et al. (EP 0070685 and Heller and Jablonski, U.S. Pat. No. 4,996,143 (issued Feb. 26, 1991)). In this method, hybridization probes are designed to provide predetermined nucleotide base unit spacings between the donor and acceptor fluorophores. When the probes are hybridized to the target polynucleotide the fluorophores paired for non-radiative energy transfer are optimally separated by 2 to 7 nucleotide base units.
FRET has also been used extensively to measure conformational changes in RNA (for review see Yang and Millar, 1997) including studies of the overall geometry of four-way RNA junctions (Duckett et al., 1995), the hammerhead ribozyme (Tuschl et al., 1994; Bassi et al., 1997) and the kinking RNA helices by bulged nucleotides (Gohlke et al., 1994). These studies have taken advantage of the ability of FRET to measure changes in distances between two probes. In each of these applications the nucleic acid under study is assembled from several chains, two of which have been site-specifically labeled with fluorescent probes. Changes in the folding of the assembled nucleic acid complexes changes the distances between the fluorescent probes and alters the intensity of the fluorescent emission spectra.
Fluorescence energy transfer can also measure conformational changes in DNA induced by proteins. For example, (Bazemore et al., 1997) measured RecAcatalyzed pairing and strand exchange in solution by energy transfer between fluorescent dyes placed at the ends of DNA oligonucleotides. RecA induced pairing of a single-stranded DNA molecule with a DNA duplex increased the energy transfer, whereas strand displacement resulted in a decrease in energy transfer.
There have been comparatively few attempts to use fluorescence methods to measure the formation of protein-DNA complexes. Drees et al., (1996) developed a protein-DNA interaction assay based on the ability to label heat shock protein with fluorescein. Upon binding to DNA the heat shock protein forms a trimer and this is accompanied by an increase in fluorescence. To measure the stoichiometry of the heat shock protein-DNA complex, complexes were formed between the fluorescently labeled heat shock protein and thiazole orange thioazole blue heterodimer (TOTAB) labeled-DNA. The ratios of the protein and DNA determined by two-color fluorescence emission assay. Measurement of complex formation by FRET was not attempted in these experiments.
6. Use of Fluorescence to Measure Ligand Binding to RNA
An improvement in the technology for measuring the ability of small molecules to bind to RNA is to utilize fluorescent reporters. Current methods all rely on the labeling of either the nucleic acid or the ligand with a fluorescent tag and measuring changes in fluorescence emission spectrum after binding. For example, Royer (U.S. Pat. No. 5,445,935 (issued Aug. 29, 1995)) described the use of polarization of the fluorescence emission from a labeled macromolecule, such as a DNA or RNA oligonucleotide, to assess the binding of the labeled macromolecule to a second unlabeled macromolecule, such as a protein. Similarly, (Metzger et al. (1997) have measured binding of unlabeled peptides derived from Tat to TAR RNA by measuring quenching of the intrinsic fluorescence of the peptide after it is bound to RNA.
In another application of fluorescence polarization, Richardson and Schulman (U.S. Pat. No. 4,257,774 (issued Mar. 24, 1981)) reported a method for detecting compounds that interact with nucleic acids by inhibition of acridine orange binding to the nucleic acid which, results in a change in fluorescence polarization. This method is of limited practical use because the binding of acridine is through intercalation at a wide variety of sites on double-helical structures, with the consequent result that the specificity of the assay is limited.
Wang and Rando (U.S. Pat. No. 5,593,835 (issued Jan. 14, 1997) and Wang et al., 1997) have discovered that the attachment of certain fluorescent moieties to an aminoglycoside antibiotic enables the subsequent binding interaction of the antibiotic with an RNA molecule to be enhanced. They have used this property to develop quantitative screening methods and kits for RNA binding compounds. In their method the fluorescently-labeled antibiotic is bound to a pre-selected region of the target RNA, thereby forming a complex which is less fluorescent that the unbound fluorescent antibiotic because of quenching of the fluorescent moiety due to its interaction with the target RNA molecule. The complex is then mixed with a compound-to-be-tested, and the fluorescence of the antibiotic measured. The antibiotic becomes more fluorescent if the compound displaces the antibiotic in the complex and binds to the pre-selected region of the target RNA.
A limitation to the use of aminoglycosides as fluorescent reporters is that these compounds are known to interact with a wide range of cellular and viral RNAs with similar affinities. For example, aminoglycosides are able to inhibit the self-cleavage activity of the hepatitis delta virus (HDV) ribozyme (Rogers et al., 1996), as well as group I self-splicing introns and the so-called xe2x80x98hammerheadxe2x80x99 ribozyme (von Ahsen and Schroeder, 1991; Wank et al., 1994; Rogers et al., 1996). Furthermore, neomycin is able to inhibit HIV-1 Rev binding to its target RRE RNA (Zapp et al., 1993; Werstuck et al., 1996). Similarly, Wang et al., (1997) compared the binding of various aminoglycosides to RRE RNA and ribosomal RNA with the goal of quantitatively determining the nature of the binding interactions between aminoglycoside antibiotics and biologically relevant RNA targets. This study concluded that the specificity for natural RNA constructs in binding aminoglycosides is very limited; not only can aminoglycosides bind many RNA structures with similar affinity, but large families of aminoglycosides are generally active as antibiotics, suggesting that inherent specificity for a particular aminoglycoside is limited (Wang et al., 1997).
In certain circumstances a fluorescently-labeled peptide has been used in place of an antibiotic to bind RNA. Wang et al. (1997) have prepared a fluorescent analogue of Rev34-50 (Fl-Rev34-50) and showed by fluorescence anisotropy (polarization) measurements that this peptidic-compound can bind the HIV-1 RRE region. Binding of unlabeled aminoglycoside antibiotics to the RRE can be measured in competition to the binding of the Fl-Rev34-50. As in the previous assay using antibiotics, quenching of the fluorescent group is due to its binding to the RNA target itself.
A second, and more general, limitation to the use of a single fluorescent group on a reporter molecule is that this group has to interact directly with the RNA target in order to show alterations in its fluorescence emission spectrum. This severely limits the number of positions on the reporter that can be modified and also alters the nature of the binding of the reporter to RNA.
Laing et al., (WO98/39484) have developed a screening method for compound binding to RNA based on the measurement of conformation changes in the target RNA. The RNA conformational changes are detected by the binding a fluorescently labeled probe, typically a fluorescently-labeled oligonucleotide which is complementary to part of the target RNA sequence. In this method the target RNA is unlabeled and interactions between the probe and the target RNA sequence are detected by fluorescence anisotropy. Binding of a test compound is detected by inhibition of the interactions between the oligonucleotide probe and the target RNA sequence.
Arenas et al. (WO97/09342) have described high-throughput screening methods for compounds that bind RNA. The Arenas et al. method is based on measuring changes in the conformation of an RNA target in the presence of a pre-defined ligand and in the presence or absence of test compounds. The method is based on the ability of certain test ligands, typically oligonucleotides, to form complexes with partially unfolded RNA targets and alter their folded states (i.e. its native conformation as defined by its particular patterns of intramolecular base-pairing and higher order structures). In the Arenas et al. method, experimental conditions are chosen so that the target RNA is subjected to unfolding (i.e., disruption of Watson-Crick base pairs) in the presence of the ligand. If the test ligand binds to the unfolded form of the target RNA under these conditions, then the relative amount of folded RNA can be compared to the relative amount of the complex formed between the target RNA and the ligand. The compounds that bind to the native conformation of the target RNA can then be detected as an increase in the proportion of folded target RNA in the sample compared to the amount of unfolded RNA-ligand complex.
In certain embodiments of the Arenas et al. method, FRET is used to detect either the folded target RNA or the unfolded RNA-ligand complex. In one such embodiment, the ligand is a fluorescently labeled oligonucleotide which is complementary to the target RNA, and two fluorescent groups come into proximity after the unfolding of the target RNA and the formation of a complex between the unfolded RNA and the oligonucleotide ligand.
It is an object of the invention to apply FRET methodologies to measure the formation of a complex between a fluorescently-labeled reporter molecule and fluorescently-labeled RNA target. One reason why this approach has not been undertaken previously is that NMR studies have shown that of RNA-peptide complexes are in intermediate exchange, suggesting a high degree of conformational flexibility and dynamic exchange at the RNA-peptide interface (Puglisi et al., 1992; Aboul-ela et al., 1995; Brodsky and Williamson, 1997; Cai et al., 1998; De Guzman et al., 1998). These dynamic properties are, in theory, a severe hindrance to the development of FRET.
The invention provides a method for determining whether a test compound binds to a target RNA, the method comprising the steps of: (a) contacting the test compound with a pair of indicator molecules comprising a reporter labeled with a donor group or an acceptor group and the target RNA labeled with a complementary acceptor or donor group, the pair being capable of binding to each other in an orientation that permits the donor group to come into sufficient proximity to the acceptor group to permit fluorescent resonance energy transfer and/or quenching to take place; and (b) measuring the fluorescence of the target RNA and/or the reporter molecule in the presence of the test compound and comparing this value to the fluorescence of a standard.
In preferred embodiments, the standard comprises the indicator pair in the presence or absence of test compound, the fluorescently-labelled target RNA in the presence or absence of test compound, or fluorescently-labelled reporter molecule in the presence or absence of test compound. It will be appreciated that the fluorescence of the standard may have been determined before performing the method, or may be determined during or after the method has been performed. It may be an absolute standard.
The method may also be used in the identification of compounds that bind to the target RNA from within a plurality of test compounds, such as in screening methods. The method may, therefore, involve the initial step of providing a plurality of test compounds, which may include compounds not already known to bind to the target RNA sequence.
In a typical embodiment, therefore, the invention provides a method of screening for compounds that bind to a target RNA, comprising the steps of (a) contacting a test compound with an indicator complex, the indicator complex comprising a fluorescently-labeled reporter molecule bound to a fluorescently labeled target RNA in an orientation that permits the fluorescent groups present on each molecule to come into sufficient proximity to permit fluorescent resonance energy transfer to take place; and (b) measuring the fluorescence of the target RNA and the reporter molecule in the presence of the test compound and comparing this value to the fluorescence of a standard.
In preferred embodiments of the methods of the invention, the reporter molecule comprises a peptide, a protein, a lipid, a polysaccharide, or a small organic molecule. In other preferred embodiments, the reporter comprises a linear peptide or derivative thereof, a cyclic peptide or derivative thereof, a linear or cyclic peptoid or derivative thereof, or a peptidomimetic analogue. A reporter molecule also may comprise an oligonucleotide, or derivative thereof, that is able to form a complex with the target RNA under conditions where the RNA is not subject to unfolding (for example, it forms a complex with the target RNA by forming triple helices with the folded form of the target RNA).
Typically, the reporter binds the target RNA with a Kd of between 1xc3x9710xe2x88x9212 and 1xc3x9710xe2x88x924 M, and the target RNA is between 5 and about 500 nucleotides in length.
In other embodiments, the target RNA is derived from fungal, viral, bacterial, or eukaryotic RNA, and may be chemically modified.
In a particularly preferred embodiment, the target RNA is a viral RNA from a region of the TAR of HIV.
In certain embodiments of the invention, the target RNA and reporter are selected from the following pairs:
Preferably, the target RNA is a viral RNA from a region of the RRE of HIV.
In certain other embodiments, the target RNA and reporter are selected from the following pairs:
In other preferred embodiments, the target RNA and the reporter molecule are fluorescently labelled by covalent attachment of a fluorescent group. For instance, the target RNA may be fluorescently labelled at the 3xe2x80x2 or 5xe2x80x2 end of a strand within the target RNA, or within the chain of the target RNA.
It also may be preferred in some instances that the reporter molecule or the target RNA molecule is adhered to a solid support.
The invention also includes a method for determining the presence in a biological sample of a compound that binds to a target RNA molecule, comprising (a) contacting the sample with a pair of indicator molecules comprising a reporter labelled with a donor group or an acceptor group and the target RNA labelled with a complementary acceptor or donor group, the pair being capable of binding to each other in an orientation that permits the donor group to come into sufficient proximity to the acceptor group to permit fluorescent resonance energy transfer and/or quenching to take place; and (b) measuring the fluorescence of the target RNA and the reporter molecule as an indication of binding. Preferably in this method, said biological sample comprises a tissue or fluid from a mammal.
In the methods of the invention, it also is preferred that either (i) the donor is attached to the target RNA, and the acceptor is attached to the reporter molecule, or (ii) the donor is attached to the reporter molecule, and the acceptor is attached to the target RNA.
As used herein, the term xe2x80x9cdonorxe2x80x9d refers to a fluorophore which absorbs at a first wavelength and emits at a second, longer wavelength. The term xe2x80x9cacceptorxe2x80x9d refers to a fluorophore, chromophore or quencher with an absorption spectrum which overlaps the donor""s emission spectrum and is able to absorb some or most of the emitted energy from the donor when it is near the donor group (typically between 1-100 nm). If the acceptor is a fluorophore capable of exhibiting FRET, it then re-emits at a third, still longer wavelength; if it is a chromophore or quencher, then it releases the energy absorbed from the donor without emitting a photon. Although the acceptor""s absorption spectrum overlaps the donor""s emission spectrum when the two groups are in proximity, this need not be the case for the spectra of the molecules when free in solution. Acceptors thus include fluorophores, chromophores or quenchers that, following attachment to either the RNA target molecule or to the reporter molecule, show alterations in absorption spectrum which permit the group to exhibit either FRET or quenching when placed in proximity to the donor through the binding interactions of two molecules.
As used herein, references to xe2x80x9cfluorescencexe2x80x9d or xe2x80x9cfluorescent groupsxe2x80x9d or xe2x80x9cfluorophoresxe2x80x9d include luminescence and luminescent groups, respectively.
In other preferred embodiments, the acceptor is able to quench the fluorescence of the donor after binding of the target RNA and the reporter.
As used herein, the term xe2x80x9cquenchingxe2x80x9d refers to the transfer of energy from donor to acceptor which is associated with a reduction of the intensity of the fluorescence exhibited by the donor. In certain preferred embodiments of the invention, only quenching of the donor due to the proximity of the acceptor in the reporter/RNA complex is measured. In certain embodiments of the invention, the target RNA carries a chromophore or fluorophore that quenches the fluorescence of the fluorescent group on the reporter after binding of the two molecules. In other embodiments of the invention, the reporter carries a chromophore or fluorophore that quenches the fluorescence of the fluorescent group on the target RNA after binding of the two molecules.
In some methods according to the invention, the target RNA, the reporter, and the test compound are mixed, and the fluorescence of the mixture is compared to standards. In other methods, the test compound is first mixed with the labelled RNA in order to form a complex in the absence of the labelled reporter, and the reporter is then added. Alternatively, a complex is pre-formed between the labelled RNA and the labelled reporter molecule before addition of the test compound.
The invention also encompasses a kit for determing whether a test compound binds to a target RNA, the kit comprising (a) a target RNA labelled with a donor group or an acceptor group and (b) a reporter labelled with a complementary acceptor or donor group, wherein the reporter and the target RNA are capable of binding to each other in an orientation that permits the donor group to come into sufficient proximity to the acceptor group to permit fluorescent resonance energy transfer and/or quenching.
Further features and advantages of the invention will become more fully apparent in the following description of the embodiments and drawings thereof, and from the claims.